CN101338279A - Hyphomycetes mite-killing preparation - Google Patents
Hyphomycetes mite-killing preparation Download PDFInfo
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- CN101338279A CN101338279A CNA2008100615759A CN200810061575A CN101338279A CN 101338279 A CN101338279 A CN 101338279A CN A2008100615759 A CNA2008100615759 A CN A2008100615759A CN 200810061575 A CN200810061575 A CN 200810061575A CN 101338279 A CN101338279 A CN 101338279A
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- Agricultural Chemicals And Associated Chemicals (AREA)
Abstract
The invention discloses a high efficiency capillitium fungi acaricide which can effectively control phytophagous harmful mites, reduce the usage of chemical pesticides and improve the safety of agricultural products. The scarab Metarrhizium anisopliae ZJUIM24-562S strain and sorosphere Beauveria bassiana ZJUIM17-340 strain of the invention are preserved on February 25, 2008 and the preserve codes separately are CGMCC No.2375 and CGMCC No.2374. The preparation comprises capillitium fungi conidium, mineral oil, emulsifying agent, uv-protector and stabilizer. The results of indoor and outdoor tests show that capillitium fungi acaricide has ideal extermination activity and field control efficiency for various phytophagous harmful mites. Therefore, the high efficiency capillitium fungi acaricide will fill the study knowledge and technical gaps of fungi acaricide in China.
Description
Technical field
The present invention relates to the Hyphomycetes mite-killing preparation of one class preventing and control phytophagy evil mite.
Background technology
The kind of tetranychid general reference Tetranychidae belongs to phytophagous mites, is generally polyphagy crop and economic plants evil mite, and kind is many, and it is wide to distribute.Tetranychid kind in China's cotton region harm mainly contains 5 kinds of carmine spider mite, truncate leaf mite, Tetranychus urticae, Dunhuang tetranychid and Turkestan tetranychids etc.The most serious phytophagy tetranychid of harm is a citrus red mite (crm) in vast citrus garden, and obviously rising in recent years becomes the citrus primary pest.Except that citrus, also endanger plants such as pears, peach, persimmon, jujube, tea, mulberry, sweet osmanthus, weeping willow, Chinese rose and Chinese ilex.Tetranychid usually with become mite, if mite and young mite draw juice at the plant blade back, the blade face moisture transpiration is strengthened, the chlorophyll variable color, photosynthesis is suppressed, thus make the blade face redden, dry up, come off in addition whole plant withered, reduce output and influence quality.For example have much representational carmine spider mite (having another name called tetranychus telarius, two-spotted spider mite etc.), can endanger more than 100 kind of field crop in the whole world and economic plants, cause the tremendous economic loss.
Tetranychid saw after the sixties in 20th century obvious harm beginnings of farm crop, be commonly considered as a large amount of use of chemical insecticide and cause increase wildness again.Since then, chemical prevention is the main means of big area mite evil control, organochlorine (as kelthane) and organotin (as the intact tin of mite) preparation is the efficient miticide of using always, but goes up disabled many crops (especially melon and fruit, vegetables, tealeaves) because of its high malicious high residue.Because special ecological strategy and the environmental compatibility of mite class, the resistance problem of mite class is more and more outstanding.Tetranychid can strengthen tens of to hundreds of times in 6～16 generations to the resistance of organochlorine acaricide; To organophosphorus, the organic nitrogen chemical insecticide is very insensitive or resistance is extremely strong, chrysanthemum ester insecticide even the procreation of its population had to help increase effect.The low toxicity and low residue pesticide imidacloprid that is widely used in common pest control at present also is found has the effect that stimulates tetranychid to lay eggs.This shows that harm of tetranychid and resistance problem are very outstanding.Problem how effectively to control the phytophagy tetranychid has become domestic and international Focal Point of Common Attention.
Hyphomycetale biocontrol fungis such as green muscardine fungus, muscardine, Paecilomyces varioti are the entomogenous fungis that a class can infect insect and mite class, are Deuteromycotina hyphomycetes fungi.Such biocontrol fungi has good adaptability to edatope, and host range is extensive, and it is ripe that production technique and formulation technology have become, thereby the more satisfactory bacterial classification of fungus insecticide research and development.Since nineteen ninety-five, the preparation series of beauveria bassiana Beauveria bassiana has successively been registered by American-European countries, as Mycotrol-ES, Mycotrol-WP, Mycotrol-O, BotaniGuard-ES, BotaniGuard-WP, CornGuard-ES, ComGuard-G, Naturalis (U.S.), Ago BiocontrolBassiana 50 (Colombia), Ostrinil (France), Beauveria Schwerzer (Sweden) and Engerlingspliz products such as (Germany), be mainly used on the various cash crop such as high added value organic vegetable melon and fruit that do not allow the residual existence of chemical pesticide of control aphid, suckings pest such as aleyrodid also are used to prevent and treat subterranean pest-insect and locust.Paecilomyces fumosoroseus also is the emphasis of having researched and developed since the external nineties, the commodity preparation that comes out comprises the preparation PFR-97 of Biocon (Holland), Pae-sin (Mexico), Ago Biocontrol Paecilomyces 50 (Colombia), Bemisin (Venezuela) and US and European development and PreFeRal etc., and they also are mainly used in suckings pest such as control aleyrodid.Also have the green muscardine fungus preparation GreenMuscle that produces in the South Africa registration, be mainly used in the big area locust control.External obtaining of these achievements in recent years mainly had benefited from the research and development input that continues to increase and inoculum production, formulation are processed and the continuous progress of application supporting technology.
In evil mite microbial control research, the minority pathogenic bacteria of directly infecting the mite class is given priority to for a long time, is newly connect mould as Tang Pusen by hair spore and Florida State.Regrettably these mite class obligate pathogenic fungies are difficult to exploitation becomes the preparation with commodity meaning, can only consider to utilize the popular naturally of them and play to a certain degree control action kou in specific crop ecological system (as the orchard), be difficult to use as normal intervention means.So more external scholars turn to the insect biocontrol fungi of several classics, wish that they also can play a role in control of pest mite.But owing to become the mite individuality very little, very active again, very difficult with the biometric techniques that efficient mite bacterial classification bacterial strain screening extremely is a purpose, relevant research report is very few, still is in the elementary exploratory stage at present.Screening obtains efficiently to kill the mite bacterial strain, and develop cost low, use the prescription and the blending process of high purity spore powder production technique and spore preparation easily, and it is then more rare to be successfully applied to the case of field control of pest mite.
Summary of the invention
The present invention at first provides two kinds of Hyphomycetes, and they are respectively: Metarhizium anisopliae ZJUIM24-562 bacterial strain (the Latin formal name used at school is Metarhizium anisopliae), its preserving number is CGMCC No.2375.Beauveria bassiana ZJUIM17-340 bacterial strain (the Latin formal name used at school is Beauveria bassiana), its preserving number is CGMCCNo.2374; Their preservation time is: on February 25th, 2008, depositary institution is China Committee for Culture Collection of Microorganisms common micro-organisms center.They are respectively the efficient high virulence Hyphomycetes bacterial strain of the phytophagy evil mite that can efficiently kill on the multiple kinds of crops.
Metarhizium anisopliae individual morphology of the present invention is characterized as: bacterium colony is cultivated on the SDAY flat board and is the fine hair shape, initial white, and olive-green when producing spore, the later stage is that greyish-green is to deep green; The mycelia tool is separated and branch, and transparent, diameter is 1.5-2.0 μ m.Conidiophore is difficult to and the mycelia difference, the about 2 μ m of diameter, and its end produces doleiform stigma, forms the conidium of long-chain usually continuously with basipetal from the end of bottle stalk.Conidium is unicellular, is oblong to cylindric, blunt section shape in two ends or blunt garden, and size variation is big, 5～9 * 2～4.5 μ m.Look bright during single the existence, olive-green when in heaps.
Beauveria bassiana individual morphology of the present invention is characterized as: bacterium colony is cultivated bacterium colony and is velvet-like on the SDAY flat board, the back side is light violet magenta; Conidiogenous cell is dense to cluster in mycelia, conidiophore or the vesicle that expands, and spherical neck obviously prolongs into thick 1 μ m, reaches the product spore axle about 20 μ m to doleiform, and the little tooth of tool is dashed forward on the axle, is geniculation; Conidium is spherical or subsphaeroidal, smooth transparent, and size is 2～3 * 2～2.5 μ m.
Another technical problem to be solved of the present invention is the above-mentioned defective that overcomes prior art, the harmful mite of phytophagy big at causing harm property in the present agriculture production, outstanding, the general effect of chemical control difference of resistance provides the Hyphomycetes mite-killing preparation that has the effect of tetranychid control efficiently, can significantly reduce chemical acaricidal practicality and have the environmental friendliness characteristics.For this reason, the present invention by the following technical solutions: it is for being the oil suspending agent of activeconstituents with the Hyphomycetes spore powder, and its external phase is mineral oil or vegetables oil.
Described Hyphomycetes spore powder can be: the high purity spore powder that adopts the two-phase zymotechniques to make by Metarhizium anisopliae CGMCC 2375 or beauveria bassiana CGMCC 2374, the spore powder spore content is greater than 1,000 hundred million/gram powder, live the spore rate greater than 90%, and water content is less than 8%; In mineral oil, can add xitix (uv-protector) and cellulose sodium carboxymethyl (stablizer) again; mix and make the fungal spore oil suspending agent; the feed ratio of described high purity spore powder and mineral oil is 100～150 grams per liters, and the feed ratio of described xitix and cellulose sodium carboxymethyl and mineral oil is respectively 1～5 grams per liter and 5～10 grams per liters.
Used carrier oil is the compound prescription of industrial paraffin oil or industrial paraffin oil in the described oil suspending agent.
Another technical problem to be solved of the present invention provides the fungal spore suspended emulsion agent that the above-mentioned Hyphomycetes mite-killing preparation of a kind of usefulness is made, it with above-mentioned Hyphomycetes mite-killing preparation and the emulsifying agent that possesses biocompatibility according to 5～10: 100 volume ratio mixes.
The agent of described biocompatibility good emulsifying can be adopted non-ionic emulsifier, such as fatty alcohol-polyoxyethylene ether and hydroxyl polyethylene oxide block copolymer mixture.But, pretend and face time spent mixed preparing processing because long-term mixed storage has certain influence to fungus conidium.
Through to described Hyphomycetes mite-killing bacterial strain to being that the indoor acaricidal activity of the phytophagy tetranychid of representative is measured with the carmine spider mite, Metarhizium anisopliae ZJUIM24-562 and beauveria bassiana ZJUIM17-340 have the very strong insecticidal activity that infects to carmine spider mite.Adopt Potter spray tower method by becoming two growth phases of mite with female from low paramount order inoculation carmine spider mite ovum, cultivate in the incubator of 25 ℃ of 12L:12D and observed 7～9 days, statistical study obtains described Metarhizium anisopliae ZJUIM24-562 bacterial strain to carmine spider mite ovum and the medial lethal dose (LC that becomes mite
50) be respectively 179 (99-325) and the individual spore/mm of 135 (75-224)
2Described beauveria bassiana ZJUIM17-340 bacterial strain is to carmine spider mite ovum and the medial lethal dose (LC that becomes mite
50) be respectively 191 (80-450) and the individual spore/mm of 42 (22-82)
2
Described Hyphomycetes mite-killing bacterial strain has excellent control mite effect in the field equally for phytophagy tetranychids such as citrus red mite and cotton red spiders.First conidial powder with described Hyphomycetes mite-killing bacterial strain is mixed with the spore suspended emulsion agent as stated above and carries out motor-driven atomizing, facility are 1.6 horsepowers of WFB-18AC type power knapsack atomizing devices, by the rated output of 5000 commentaries on classics/min, spore suspended emulsion agent concentration standard turns to 1 * 10
10Individual spore/mL, dilute with water is 100 times during use, presses that the fluid volume of 150L/ha is motor-driven decides the power operation, every spraying in 15 days 1 time, sprays altogether 2 times.Go down rate and relative control effect of cotton red spider average population after Metarhizium anisopliae ZJUIM24-562 bacterial strain is handled in 35 days reaches 80.7% and 85.8% respectively; Go down rate and relative control effect of cotton red spider average population due to the beauveria bassiana ZJUIM17-340 bacterial strain reaches 69.8% and 77.9% respectively.Under the same test method, nearly 45 days experimental observation is in the phase, and described two kinds are killed the mite bacterial strain relative control effect of citrus red mite is all reached more than 85%.
Test shows, Hyphomycetes mite-killing preparation of the present invention has the phytophagy tetranychid and to control effect preferably, can reduce chemical acaricidal use in a large number, improves the security of agricultural-food.
Embodiment
The preservation of embodiment 1, hyphomycetale biocontrol fungi and cultivation
Hyphomycetale biocontrol fungis such as Metarhizium anisopliae ZJUIM24-562 and beauveria bassiana ZJUIM17-340 are stored in-72 ℃ of Ultralow Temperature Freezers with dry conidial powder and sterilization white sand blended form.The preservation bacterial classification all activates about 7 days down in 25 ℃ on sabouraud medium SDAY flat board, be used for the production of spore powder.Wherein sabouraud medium SDAY prescription (W/W) is: glucose 4%, peptone 1%, yeast 1% and agar 2%.
The production of embodiment 2, high purity conidial powder
A kind of vertical multilayer spore-generating box (patent publication No. CN 1710059A) is adopted in the production of the high purity conidial powder of hyphomycetale biocontrol fungi, produce as liquid-solid two phase fermentation methods (patent publication No. CN 1415736) of main raw material with the low value old rice, specifically carry out according to following steps:
1) described hyphomycetale is efficiently killed the mite bacterial strain, use Sa Shi nutrient solution or wheat bran vat liquor to carry out the liquid phase fermentation, obtain mycelia liquid.
2) selecting for use low value rice to clean drains after steam sterilizing and slaking to 5～6 maturations are cooled off the back as the solid phase fermented substrate.
3) in the above-mentioned solid phase fermented substrate, sneak into the mycelia liquid of above-mentioned liquid phase fermentation gained by the ratio of 8～12 liters/100 kilogram-metres, machine stir the inoculation material.
4) will inoculate material and put into spore-generating box and carry out closed fermentation and produce spore, the temperature in the spore-generating box is controlled at 25 ± 2 ℃, relies on humidifier to keep relative humidity in the spore-generating box 〉=95%, and the inoculation material ferment in spore-generating box and reflected 72～96 hours.
5) closing humidifier, is that temperature is 25 ± 2 ℃ in the spore-generating box maintenance sealed state control spore-generating box, continues fermentation reaction 72～120 hours, gets fermentation materials;
6) surface covers with the described fermentation materials that kills mite bacterial strain spore powder being no more than 35 ℃ of following air seasonings to water content below 40%, receives spore machine results spore powder with the MK-V type.The spore powder spore content that obtains reaches 1,000 hundred million/gram, and the spore rate of living is greater than 90%.After the normal temperature vacuum was drained, the water content of spore powder was less than 8% again.
The acaricidal activity of embodiment 3, hyphomycetale biocontrol fungi is measured
The high purity spore powder that obtains among the embodiment 2 is become high, medium and low three spore dosage spore suspensions with 0.02%Tween 80 solution dilutions, is blank with the aqueous solution that only contains 0.02%Tween 80.Test adopts Potter spray tower method to press from low paramount order, at 0.7kg/cm
2Pressure under the spore fluid suspension of 2ml is sprayed on the blade that carries full female one-tenth mite of tetranychid or mite ovum of Stage microscope of diameter 11cm.(20cm * 20cm) collect spore, cotton blue dyeing back is at 5 samplings of microscopically microscopy (0.2165mm with lying against the other cover glass of blade for actual dosage of inoculation under each concentration
2/ the visual field) spore count is standardized as spore count/mm thereby will fall the spore adhesion amount that is deposited on mite body and the blade thereof
2Place the incubator of 25 ℃ of 12L:12D to cultivate observation 7～9 days female one-tenth mite or mite ovum after the inoculation, the data that obtain are carried out statistical study, calculate to obtain for the kill activity of examination biocontrol fungi to female one-tenth mite of carmine spider mite and mite ovum.Wherein, described Metarhizium anisopliae ZJUIM24-562 bacterial strain is to carmine spider mite ovum and the medial lethal dose (LC that becomes mite
50) be respectively 179 (99-325) and the individual spore/mm of 135 (75-224)
2Described beauveria bassiana ZJUIM17-340 bacterial strain is to carmine spider mite ovum and the medial lethal dose (LC that becomes mite
50) be respectively 191 (80-450) and the individual spore/mm of 42 (22-82)
2
The preparation of embodiment 4, Hyphomycetes mite-killing bacterial strain spore oil suspending agent
Adopt commercially available industrial paraffin oil or industrial paraffin oil mixture as the inert liquid phase medium, add preparedly among the embodiment 2 efficiently kill mite Hyphomycetes bacterial strain high purity conidial powder, as the cellulose sodium carboxymethyl of stablizer with as the xitix of uv-protector.Wherein the feed ratio of high purity spore powder and mineral oil is 100～150 grams per liters, the feed ratio of cellulose sodium carboxymethyl and mineral oil is 1～5 grams per liter, the feed ratio of xitix and mineral oil is 1～5 grams per liter also, promptly makes the spore oil suspending agent of Hyphomycetes mite-killing bacterial strain after mixing.Used industrial paraffin oil helps the storage of microbial inoculum through the long storage test determination, and biocompatibility is good.
The Metarhizium anisopliae ZJUIM24-562 of preparation and effective spore content of beauveria bassiana ZJUIM17-340 spore oil suspending agent are not less than 10,000,000,000/milliliter, and water content is no more than 2%.The Metarhizium anisopliae spore oil suspending agent that mixes is blackish green or near black liquor, and beauveria bassiana spore oil suspending agent is faint yellow or khaki color.
The preparation of embodiment 5, Hyphomycetes mite-killing bacterial strain spore suspended emulsion agent
With embodiment 4 prepared spore oil suspending agents and good fatty alcohol-polyoxyethylene ether of biocompatibility and hydroxyl polyethylene oxide block copolymer mixture, according to 5～10: 100 volume ratio is mixed, and stirs and promptly makes Metarhizium anisopliae and beauveria bassiana spore suspended emulsion agent.
Fungi kills mite spore suspended emulsion agent can carry out conventional capacity (500～1000 times of diluents) or lower volume (100～150 times of diluents) spraying by dilute with water.
Embodiment 6, Hyphomycetes mite-killing bacterial strain spore suspended emulsion agent are at the application and the miticidal effect in field
1) Xinjiang cotton red spider control test
Select two heavier cotton fields of mite evil to carry out the field control effectiveness test of cotton red spider in cotton region, Takla Makan Desert the northern fringe stage casing, Alar City, Xinjiang in July, 2006 to August.Test is with 100～150 times of motor-driven sprayings of carrying out lower volume of embodiment 5 prepared spore suspended emulsion agent dilute with waters.Bacteria using amount in the diluent is: make its spore oil suspending agent consumption that includes reach 1.5～2.3 liters/hectare.
Intertrial interval 10～15 days is continuously with bacterium 2 times, can both control two-spotted spider mite causing harm to cotton effectively.Kill the mite bacterial strain with 5～10 days described two strain hyphomycetales behind the bacterium relative control effect of two-spotted spider mite has all been reached more than 70%, for the second time with then being stabilized between 75%～95% behind the bacterium.
2) Zhejiang citrus red mite control test
Choose representative tangerine garden in October, 2007 to November in Quzhou City and Taizhou liang citrus main producing region, Zhejiang and carried out 45 days citrus red mite field control effectiveness test by a definite date.Test is same with 100～150 times of motor-driven sprayings of carrying out lower volume of embodiment 5 prepared spore suspended emulsion agent dilute with waters.
Test was also used bacterium 2 times in 10～15 days at interval continuously, found that described Hyphomycetes mite-killing preparation is remarkable to the field control effect of citrus red mite, controlled the harm of citrus red mite to citrus effectively.The 5th day relative control effect is 64.5%～72.7% and 58.9%～72.2% after Quzhou and two places, Taizhou are with bacterium, and reached respectively more than 85% and 90% to the 2nd relative control effect with two places in behind the bacterium nearly 30 days, be significantly higher than the prevention effect of chemical pesticide pyridaben.And the lasting period of microbial inoculum is very long, by the sign that still not have population density significantly to rebound to March of next year, show described Hyphomycetes mite-killing preparation to stride seasonal effect remarkable.
Claims (10)
1, Metarhizium anisopliae ZJUIM24-562 bacterial strain (Metarhizium anisopliae), its preserving number is CGMCC No.2375.
2, beauveria bassiana ZJUIM17-340 bacterial strain (Beauveria bassiana), its preserving number is CGMCCNo.2374.
3, Hyphomycetes mite-killing preparation is characterized in that it is is the oil suspending agent of activeconstituents with the Hyphomycetes spore powder, and its external phase is mineral oil or vegetables oil.
4, Hyphomycetes mite-killing preparation as claimed in claim 3, the feed ratio that it is characterized in that spore powder and external phase is 100～150 grams per liters.
5, Hyphomycetes mite-killing preparation as claimed in claim 3 is characterized in that adding has xitix as uv-protector in the described oil suspending agent, and the feed ratio of xitix and external phase is respectively 1～5 grams per liter.
6, Hyphomycetes mite-killing preparation as claimed in claim 3 is characterized in that adding has cellulose sodium carboxymethyl as stablizer in the described oil suspending agent, and the feed ratio of cellulose sodium carboxymethyl and external phase is respectively 5～10 grams per liters.
7, Hyphomycetes mite-killing preparation as claimed in claim 3 is characterized in that described Hyphomycetes is the described Metarhizium anisopliae ZJUIM24-562 of claim 1 bacterial strain.
8, Hyphomycetes mite-killing preparation as claimed in claim 3 is characterized in that described Hyphomycetes is the described beauveria bassiana ZJUIM17-340 of claim 1 bacterial strain.
9, a kind of fungal spore suspended emulsion agent made from the described Hyphomycetes mite-killing preparation of claim 3, it is characterized in that it with the described Hyphomycetes mite-killing preparation of claim 3 and the emulsifying agent that possesses biocompatibility according to 5～10: 100 volume ratio mixes.
10, fungal spore suspended emulsion agent as claimed in claim 9 is characterized in that the described emulsifying agent that possesses biocompatibility is fatty alcohol-polyoxyethylene ether and hydroxyl polyethylene oxide block copolymer mixture.
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